[5] Measurement of enzyme activity

This chapter describes the development of methods for the assay of enzyme activity in a cell lysate or in a partially purified enzyme preparation. These assays are also applicable during purification and for purified enzymes as well. The first component of an assay is the reaction mixture. The reaction mixture usually contains the buffer used to establish the correct pH, the substrate, and any cofactors that may be required for catalysis. The preparation of reaction mixtures involves mixing these ingredients in a reaction vessel, such as a test tube or, for some assay methods, a cuvette. The second part of an assay is initiation/incubation. A reaction is often started by the addition of the enzyme preparation to the substrate already present in the reaction mixture. This combination initiates the incubation phase and all subsequent time points are referenced to this time as zero. As the radiochemical detector cannot differentiate the free radioactive amino acid used as the substrate from that bound covalently to the tRNA, the free and the bound amino acids must be separated prior to the detection or quantitation step.